U.S. patent application number 13/476098 was filed with the patent office on 2013-02-07 for inhibitors of akt/pkb with anti-tumor activity.
This patent application is currently assigned to University of South Florida. The applicant listed for this patent is Jin Q. Cheng, Said M. Sebti, Mei Sun. Invention is credited to Jin Q. Cheng, Said M. Sebti, Mei Sun.
Application Number | 20130034598 13/476098 |
Document ID | / |
Family ID | 40229514 |
Filed Date | 2013-02-07 |
United States Patent
Application |
20130034598 |
Kind Code |
A1 |
Cheng; Jin Q. ; et
al. |
February 7, 2013 |
INHIBITORS OF AKT/PKB WITH ANTI-TUMOR ACTIVITY
Abstract
The subject invention concerns materials and methods for
inhibiting the Akt/PKB pathway. In one embodiment, a compound of
the invention inhibits kinase activity and/or phosphorylation
levels of Akt proteins. The subject invention also concerns methods
for inhibiting or killing a cancer cell or other cell in which
expression of an Akt protein is elevated or constitutively active,
comprising contacting the cell with an effective amount of a
compound of formula I. The subject invention also concerns methods
for treating cancer or a tumor in a person or animal comprising
administering an effective amount of a compound of formula I to the
person or animal.
Inventors: |
Cheng; Jin Q.; (Tampa,
FL) ; Sun; Mei; (Tampa, FL) ; Sebti; Said
M.; (Tampa, FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cheng; Jin Q.
Sun; Mei
Sebti; Said M. |
Tampa
Tampa
Tampa |
FL
FL
FL |
US
US
US |
|
|
Assignee: |
University of South Florida
|
Family ID: |
40229514 |
Appl. No.: |
13/476098 |
Filed: |
May 21, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12172831 |
Jul 14, 2008 |
8183249 |
|
|
13476098 |
|
|
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|
60949365 |
Jul 12, 2007 |
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Current U.S.
Class: |
424/450 ;
424/133.1; 424/649; 424/94.6; 435/366; 435/375; 514/19.3; 514/27;
514/49; 536/27.13 |
Current CPC
Class: |
C07H 19/23 20130101;
A61P 9/10 20180101; C07D 471/04 20130101; A61P 35/00 20180101; A61P
5/32 20180101 |
Class at
Publication: |
424/450 ;
536/27.13; 514/49; 424/649; 514/19.3; 514/27; 424/94.6; 424/133.1;
435/375; 435/366 |
International
Class: |
A61K 31/7064 20060101
A61K031/7064; A61P 35/00 20060101 A61P035/00; A61K 33/24 20060101
A61K033/24; A61K 38/14 20060101 A61K038/14; C12N 5/09 20100101
C12N005/09; A61K 9/127 20060101 A61K009/127; A61K 39/395 20060101
A61K039/395; A61P 9/10 20060101 A61P009/10; A61P 5/32 20060101
A61P005/32; C12N 5/071 20100101 C12N005/071; C07H 19/23 20060101
C07H019/23; A61K 38/50 20060101 A61K038/50 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The subject matter of this application has been supported by
a research grants from the National Institutes of Health--National
Cancer Institute and ARMY/MRMC under grant numbers R01CA107078,
DAMD17-02-1-0671, and W81XWH-05-1-0021. Accordingly, the government
has certain rights in this invention.
Claims
1. A compound of formula I: ##STR00004## wherein X is,
independently, O, N, or S; Y is O, N, or S; R.sup.1 is,
independently, --H, --OH, --NH.sub.2, --NO.sub.2, halogen, alkyl
optionally substituted with --OH, or alkoxy optionally substituted
with --OH; R.sup.2 is --H, --OH, --NH.sub.2, --C(O)NH.sub.2, alkyl,
or alkoxy, any of which can be optionally substituted with --OH,
halogen, alkyl, or alkoxy; R.sup.3 is --H, --OH, --NH.sub.2,
--C(O)NH.sub.2, alkyl, or alkoxy, any of which can be optionally
substituted with --OH, halogen, alkyl, or alkoxy; or a
pharmaceutically acceptable salt thereof.
2. The compound of claim 1, wherein each X is N or Y is O.
3. The compound of claim 1, wherein each R.sup.1 is independently
--OH or --CH.sub.2OH.
4. The compound of claim 1, wherein R.sup.2 is --NH.sub.2
optionally substituted with --OH.
5. The compound of claim 1, wherein R.sup.2 is --C(O)NH.sub.2.
6. The compound of claim 1, wherein the compound has the structure:
##STR00005##
7. A composition comprising a compound according to claim 1.
8. The composition according to claim 7, wherein said composition
comprises a pharmaceutically acceptable carrier or diluent.
9. The composition according to claim 7, wherein said composition
comprises one or more anti-cancer agents.
10. The composition according to claim 9, wherein said anti-cancer
agent is altretamine, bleomycin, bortezomib (VELCADE), busulphan,
calcium folinate, capecitabine, carboplatin, carmustine,
chlorambucil, cisplatin, cladribine, crisantaspase,
cyclophosphamide, cytarabine, dacarbazine, dactinomycin,
daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide,
fludarabine, fluorouracil, gefitinih (TRESS A), gemcitabinc,
hydroxyurea, idarubicin, ifosfamide imatinib (GLEEVEC), irinotecan,
liposomal doxorubicin, lomustine, melphalan, mercaptopurine,
methotrexate, ruitomycin, mitoxantrone, oxaliplatin, paclitaxel,
pentostatin, procarbazine, raltitrexed, streptozocin,
tegafur-uracil, temozolomide, thiotepa, tioguanine/thioguanine,
topotecan, treosulfan, vinblastine, vincristine, vindesine,
vinorelbine, melphalan, alemtuzumab, cetuximab (ERBITUX),
gemtuzumab, iodine 131 tositumomab, rituximab, or trastuzamab
(HERCEPTIN).
11. The composition according to claim 7, wherein said composition
comprises one or more of a mitotic inhibitor, an alkylating agent,
an antimetabolite, a DNA intercalator, a topoisomerase inhibitor,
an antiangiogenic agent, or an antiestrogen.
12. A method for inhibiting the survival or proliferation of a cell
or killing a cell having elevated or constitutively active
expression of an Akt protein, said method comprising contacting
said cell with an effective amount of a compound according to claim
1 or a composition comprising said compound.
13. The method according to claim 12, wherein the cell is a human
cell.
14. The method according to claim 12, wherein the cell is a cancer
or tumor cell.
15. The method according to claim 14, wherein the cancer cell is a
cancer cell of the anus, bile duct, bladder, bone, bone marrow,
bowel (including colon and rectum), breast, eye, gall bladder,
kidney, mouth, larynx, esophagus, stomach, testis, cervix, head,
neck, ovary, lung, mesothelioma, neuroendocrine, penis, skin,
spinal cord, thyroid, vagina, vulva, uterus, liver, muscle,
pancreas, prostate, blood cells (including lymphocytes and other
immune system cells), or brain.
16. A method for treating an oncological disorder in a person or
animal, said method comprising administering a therapeutically
effective amount of a compound according to claim 1 or a
composition comprising said compound.
17. The method according to claim 16, wherein said oncological
disorder is a cancer and/or tumor of the anus, bile duct, bladder,
bone, bone marrow, bowel (including colon and rectum), breast, eye,
gall bladder, kidney, mouth, larynx, esophagus, stomach, testis,
cervix, head, neck, ovary, lung, mesothelioma, neuroendocrine,
penis, skin, spinal cord, thyroid, vagina, vulva, uterus, liver,
muscle, pancreas, prostate, blood cells (including lymphocytes and
other immune system cells), or brain.
18. The method according to claim 16, wherein said ontological
disorder is associated with or characterized by cells that have
elevated or constitutively active expression of an Akt protein.
19. The method according to claim 16, wherein said method further
comprises identifying a person or animal having or who is in need
of treatment of an ontological disorder.
20. A method for inhibiting the Akt/PKB pathway in a cell, said
method comprising contacting the cell with an effective amount of a
compound according to claim 1 or a composition comprising said
compound.
21. The method according to claim 20, wherein said cell
constitutively expresses an Akt protein or said cell expresses
elevated levels of an Akt protein.
22. The method according to claim 20, wherein said cell is a tumor
or cancer cell.
23. The method according to claim 20, wherein said cell is a human
cell.
24. A method of treating a disorder in a person or animal, wherein
said disorder is associated with constitutive, abnormal, or
elevated expression of an Akt protein in a cell, said method
comprising administering an effective amount of a compound
according to claim 1 or a composition comprising said compound.
25. The method according to claim 24, wherein said disorder is
characterized by abnormal cell proliferation, cell survival, cell
migration, and/or cell differentiation.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application claims the benefit of U.S.
Provisional Application Ser. No. 60/949,365, filed Jul. 12, 2007,
which is hereby incorporated by reference herein in its entirety,
including any figures, tables, nucleic acid sequences, amino acid
sequences, and drawings.
BACKGROUND OF THE INVENTION
[0003] Akt, also called protein kinase B, represents a subfamily of
the serine/threonine kinase. Akt was first described as the
cellular homologue of the product of the .nu.-akt oncogene
(Bellacosa et al. 1991), and it has three members, Akt1/PKB.alpha.,
Akt2/PKB.beta. and Akt3/PKB.gamma. (Cheng et al. 1992; Jones et al.
1991a; Jones et al. 1991b). Activation of Akt depends on the
integrity of the pleckstrin homology (PH) domain, which mediates
its membrane translocation, and on the phosphorylation of
Thr.sup.308 in the activation loop and Ser.sup.473 (Konishi et al.
1995). Phosphoinositides, PtdIns-3,4-P2 and PtdIns-3,4,5-P3,
produced by PI3 K bind directly to the PH domain of Akt, driving a
conformational change in the molecule, which enables the activation
loop of Akt to be phosphorylated by PDK1 at Thr.sup.308 (Datta et
al. 1999). Full activation of Akt is also associated with
phosphorylation of Ser.sup.473 within a C-terminal hydrophobic
motif (Datta et al. 1999). Although the role of PDK1 in Thr.sup.308
phosphorylation is well established, the mechanism of Ser.sup.473
phosphorylation is controversial. A number of candidate enzymes
responsible for this modification have been put forward, including
integrin-linked kinase (Persad et al. 2001), PDK1 when in a complex
with the kinase PRK2 (Wick et al. 2000), Akt itself, through
autophosphorylation (Toker et al. 2000), DNA-dependent kinase (Feng
et al. 2004), and the rictor-mTOR complex (Sarbassoy et al. 2005).
The activity of Akt is negatively regulated by tumor suppressor
PTEN, which is frequently mutated in human malignancy (Vazquez et
al. 2000). PTEN encodes a dual-specificity protein and lipid
phosphatase that reduces intracellular levels of PtdIns-3,4,5-P3 by
converting them to PtdIns-4,5-P2, thereby inhibiting the PI3K/Akt
pathway (Stambolic et al. 1998).
[0004] Akt phosphorylates and/or interacts with a number of
molecules to exert its normal cellular functions, which include
roles in cell proliferation, survival, migration and
differentiation (Cheng et al. 2001). Many lines of evidence
demonstrate that Akt is a critical player in the tumor development
and progression. In addition, aberrant hyperactivation of Akt
pathway has been detected in up to 50% all human tumors (Sun et al.
2001; Cheng et cal. 1997) and is closely associated with
chemoresistance (West et al. 2002). Therefore, Akt has been an
attracting target for anti-cancer drug discovery (West et al.
2002).
[0005] In the last several years, through combinatorial chemistry,
high-throughput and virtual screening, and traditional medicinal
chemistry, a dozen inhibitors of the Aid pathway have been
identified. Lipid-based inhibitors of Akt were the first to be
developed, including perifosine (Kondapaka et al. 2003), PX-316
(Meuillet et al. 2004) and phosphatidylinositol ether lipid
analogues (Castillo et al. 2004), which were designed to interact
with the PH domain of Akt. In addition, several Akt antagonists
have been identified using high-throughput screening of chemical
libraries and rational design. These inhibitors include
9-methoxy-2-methylellipticinium acetate (Jin et al. 2004), the
indazole-pyridine A-443654 (Luo et cd. 2005), isoform-specific
allosteric kinase inhibitors (Lindsley et al. 2005) and Akt/PKB
signaling inhibitor-2 (API-2), also called triciribine/TCN (Yang et
al. 2004). APT-2/TCN is a tricyclic nucleoside that previously
showed antitumor activity in phase I and phase II trials conducted,
but multiple toxicities, including hepatotoxicity, hyperglycemia,
thrombocytopenia, and hypertriglyceridemia, precluded further
development (Feun et al. 1993; Hoffman et al. 1996). By screen of
the NCl diversity set, we have previously shown that API-2 inhibit
Akt kinase activity and stimulate apoptosis of xenografts of human
cancer cells exhibiting high Akt activity (Yang et al. 2004). This
finding has provided new interest in studying this drug and raises
the possibility that lower doses may inhibit Akt and induce tumor
cell apoptosis without the previously associated side effects (Yang
et al. 2004; Cheng et al. 2005).
BRIEF SUMMARY OF THE INVENTION
[0006] The subject invention concerns compounds, compositions, and
methods for inhibiting the Akt/PKB pathway. In one embodiment, a
compound of the invention inhibits kinase activity and/or
phosphorylation levels of Akt proteins. Compounds of the invention
have the general structure shown in formula I. In a specific
embodiment, a compound of the invention (referred to herein as
API-1) has the structure:
##STR00001##
[0007] Compounds of the invention, such as API-1, inhibit Aid
signaling in human tumor cells with aberrant Akt leading to
inhibition of cell growth and induction of apoptosis. In a
xenograft nude mice model, compounds of the invention considerably
inhibit tumor growth in the cells with hyperactivated Akt but not
in the tumors with low levels of Akt.
[0008] The subject invention also concerns methods fOr inhibiting
or killing a cancer cell or other cell in which expression of an
Akt protein is elevated or constitutively active, comprising
contacting the cell with an effective amount of a compound of
formula I.
[0009] The subject invention also concerns methods for treating
cancer or a tumor in a person or animal comprising administering an
effective amount of a compound of formula I to the person or
animal.
[0010] The serine/threonine kinase Akt/PKB pathway is frequently
hyperactivated in human cancer and functions as a cardinal nodal
point for transducing extracellular and intracellular oncogenic
signals, and thus it presents a target for molecular therapeutics.
By screening the National Cancer Institute Diversity Set, a small
molecule Akt pathway inhibitor, APT (Akt/PKB signaling
inhibitor)-1, was identified. API-1 inhibits the kinase activity
and phosphorylation level of three members of Akt family. However,
it had no the effects on the activity of the upstream activators,
PI3K and PDK1. Further, the kinase activity and phosphorylation
levels of constitutively active Akt were largely inhibited by API-1
in cell culture, while it had no effect on Akt kinase activity in
vitro. API-1 is highly selective for Akt and does not inhibit the
activation of PKC, SGK, PKA, STAT3, Erk-1/2, or JNK. The inhibition
of Akt by API-1 resulted in induction of cell growth arrest and
apoptosis in human cancer cells that harbor constitutively
activated Akt. Significantly, API-1 selectively inhibited tumor
growth in nude mice of human cancer cells in which Akt is elevated
but not of those cancer cells in which it is not. These data
suggest that API-1 is an Akt pathway inhibitor with anti-tumor
activity in vitro and in vivo and could be a potential anti-cancer
agent for patients with cancer expressing hyperactivated Akt.
[0011] Akt is a major pathway regulating cancer cell survival,
growth and tumor progression. It has been well documented that
elevated levels of Akt kinase contribute to resistance to various
cancer therapies, including cell-toxic chemotherapeutic drugs and
small molecule inhibitors of Bcr-Abl (Gleevec), Her2/Neu
(Hercptin), and mTOR (rapamycin). Blocking Akt inhibits tumor
growth in the cancer cells with hyperactivated Akt and renders
cancer cells more sensitive to chemotherapy and other targeted
therapies. Combination of API-1 with other anti-tumor drugs
provides more potent anti-tumor effects.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIGS. 1A-1D are graphical representations of the
identification of API-1 as a candidate of Akt inhibitor from the
NCI Diversity Set. FIG. 1A shows the chemical structure of API-1.
FIG. 1B is a graph showing that API-1 inhibits three members of
Akt. HEK293 cells were transfected with HA-Akt1, -AKT2 and -AKT3
and treated with with anti-HA antibody. The immunoprecipitates were
subjected to in vitro kinase assay (top). Bottom panel is a Western
blot showing expression of transfected Akt1, AKT2 and AKT3 detected
with anti-HA antibody. FIG. 1C is a graph showing that API-1
inhibits phosphorylation levels of Akt in OVCAR3 cells, which
express hyperactivated Akt. The cells were treated with API-1 at
indicated concentration for 2 hours and subjected to immunoblotting
analysis with anti-phospho-Akt-S473 antibodies (top panel). Bottom
panel shows expression of total Akt. FIG. 1D is a graph showing
that API-1 did not inhibit Akt in vitro. In vitro kinase assay of
recombinant constitutively active Akt protein in a kinase buffer
containing indicated amount of API-1. Compound E, an ATP-competitor
of multiple kinase inhibitors, was used as positive control. The
experiment was repeated three times.
[0013] FIGS. 2A-2G are graphs showing that API-1 does not inhibit
PI3K, PDK1 and the closely related members of AGC kinase family.
FIG. 2A is a graph showing in vitro PI3K kinase assay. HEK293 cells
were serum-starved and treated with API-1 (10 .mu.M) or Wortmannin
(1 nM) for 30 minutes prior to EGF stimulation. Cells were lysed
and immunoprecipitated with anti-p110a antibody. The
immunoprecipitates were subjected to in vitro kinase assay using
PI-4-P as substrate. FIG. 2B is a graph showing the effect of API-1
on PDK1 activation. In vitro kinase assay was performed with PDK1
kinase kit (Upstate Biotechnology Inc) according to manufacture's
instruction in the presence of indicated compounds. FIG. 2C is a
graph showing in vitro PKA kinase assay. Recombinant PKA was
incubated in ADB buffer (Upstate Biotechnology Inc) containing
indicated inhibitors (API-1 or PKAI) and substrate Kemptide. The
kinase activity was quantified. FIG. 2D is a graph showing the
effect of API-1 on PKA, PKC and PDK kinase activity in living
cells. OVCAR3 cells were treated with indicated concentration of
API-1 for 1 hour. Cells were lysed and immunoblotted with indicated
antibodies. FIG. 2E is a graph showing in vitro SGK kinase assay.
Recombinant SGK protein was incubated with API-1 or compound E.
Kinase assay was started by adding SGK substrate peptide and
[.gamma.-.sup.32P] ATP. The kinase activity was quantified. FIG. 2F
is a graph showing the results when HEK293 cells were transfected
with HA-SGK and treated with API-1 or Wortmannin prior to EGF
stimulation. In vitro kinase was performed with HA-SGK
immunoprecipitates using histone-H2B as substrate. FIG. 2G is a
graph showing that API-1 does not inhibit phosphorylation of Erk,
p38, JNK and Stat3. OVCAR3 cells were treated with APT-1 for 3
hours and immunoblotted with indicated antibodies.
[0014] FIGS. 3A-3D are graphs demonstrating that API-1 inhibits
constitutively active Akt and its downstream targets. FIG. 3A is a
graph demonstrating that API-1 inhibits constitutively active Akt.
HEK293 cells were transfected with indicated HA-myr-Akt1,
HA-Akt1-E40K and HA-myr-Akt2. Following treatment with API-1 for 1
hour, cells were lysed and immunoprecipitated with anti-HA
antibody. The immunoprecipitates were subjected to in vitro kinase
assay (top panel) and immunoblotting with indicated antibodies
(middle and bottom panels). FIG. 3B is a graph demonstrating that
API-1 inhibits phosphorylation of Akt downstream targets. OVCAR3
cells were treated with APT-1 (10 .mu.M) for indicated times and
immunoblotted with indicated antibodies. API-1 significantly
reduces the phosphorylation levels of Akt and its downstream
targets, GSK3.beta. and S6 protein. In FIG. 3C, AKT-SI1 inhibited
phosphorylation of Akt downstream targets. In FIG. 3D, AKT-SI1 did
not interfere with mTORC1 and mTORC2 complexes.
[0015] FIGS. 4A-4G are graphs showing that API-1 inhibits Akt
activity and cell growth and induces apoptosis in human cancer
cells with elevated Akt. FIG. 4A is a Western blot showing the
results following treatment with API-1, phosphorylation levels of
Akt and PARP cleavage were detected with anti-phospho-Akt-T308 and
cleaved PARP antibodies in indicated human cancer cell lines (top
and middle panels). The blots were reprobed with anti-actin
antibody (bottom panel). FIGS. 4B and 4C are cell proliferation
assays in which indicated cell lines were treated with different
doses of API-1 for 24 hours and then analyzed with MTT assay. FIGS.
4D-4G show an apoptosis analysis in which cells were treated with
API-1 and stained with annexin V and PI and analyzed by
FACScan.
[0016] FIGS. 5A-5L are graphics demonstrating that API-1 exhibits
anti-tumor activity in cancer cell lines with elevated Akt in mouse
xenograft. FIGS. 5A-5F are photos of the control (FIG. 5A) and
API-1-treated groups (FIG. 5B) and the related graphs (FIGS.
5C-5F). Tumor cells were subcutaneously injected into nude mice
with low level of Akt cells on right side and elevated level of Akt
cells on left side. When the tumors reached an average size of
about 100-150 mm.sup.3, animals were treated with either vehicle or
10 mg/kg/day API-1 as described in "Materials and Methods."
Representation of the mice with PANC1/OVCAR3 (FIGS. 5E and 5C,
respectively), which express elevated levels of Akt, and
COLO357/OVCAR5 (FIGS. 5F and 5D, respectively), which exhibit low
levels of Akt, xenografts treated with API-1 or vehicle (A). Panel
B shows tumor growth curve with 10 mice/group. FIGS. 5G-5J show
examples of tumor size (left) and weight (right) at the end of
experiment. API-1 significantly reduced tumor weight in PANC1 and
OVCAR3 xenografts (*P.ltoreq.0.02) as compared to DMSO control.
FIGS. 5K and 5L demonstrate that API-1 inhibits Akt phosphorylation
in vivo. API-1 treated and untreated tumor specimens were lysed and
immunoblotted with indicated antibodies.
[0017] FIGS. 6A-6C show that AKT-SI1 inhibits IGF-1 induced plasma
membrane translocation of Akt. HeLa cells were transfected on
coverslips with Myc-AKT1, serum-starved overnight and then treated
with (FIG. 6C) or without (FIG. 6B) AKT-SI1 for 30 min prior to
stimulation with IGF1 for 15 minutes. Following fixation, cells
were immuno-stained with the anti-Myc monoclonal antibody, followed
by a FITC-conjugated secondary antibody to reveal the presence of
the epitope-tagged protein in the cytosol or membrane (FIG. 6B).
Cells without treatment with AKT-SI1 and IGF1 were used as control
(FIG. 6A).
[0018] FIGS. 7A-7E show that AKT-SI1 is more potent than API-2/TCN
in inhibition of Akt kinase activity, especially constitutively
active Akt. While AKT-SI1 structure shares the ribose sugar moeity
with AP1-2/TCN (FIG. 7A), the remaining portion of these 2
molecules have no chemical similarities. Nevertheless, we compared
their capability of inhibiting Akt. HEK293 cells were transfected
with wild-type Myc-AKT1 (FIGS. 7B and 7C) and constitutively active
Myc-AKT1-E17K (FIGS. 7D and 7E). Following 36 h incubation, cells
were serum starved overnight. The wild-type Akt-transfected cells
were treated with AKT-SI1 (FIGS. 7B and 7D) or API-2/TCN (FIGS. 7C
and 7E) for 30 min and subsequently stimulated with EGF for 15 min.
Immunoprecipitation was carried out with anti-Myc antibody and the
immunoprecipitates were subjected to in vitro kinase assay using
Histone H.sub.2B as substrate (top). Inhibition of Akt kinase
activity by AKT-SI1 and API-2/TCN was quantified and calculated as
relative activity (middle). Western blot analysis shows the
immunoprecipitated AKT1 proteins (bottom panels). The experiments
were repeated three times.
[0019] FIGS. 8A and 8B show AKT-SI1 and API-2/TCN inhibit
phospho-Akt levels. Wild-type Myc-AKT1 and constitutively active
Myc-AKT1-E17K transfected HEK 293 cells were treated with the
indicated reagents and immunoblotted with anti-phospho-Akt-S473
(top) and -Myc (bottom) antibodies.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The subject invention concerns compounds and compositions
that inhibit the Akt/PKB pathway. In one embodiment, a compound of
the invention inhibits kinase activity and/or phosphorylation
levels of Akt proteins. Compounds of the invention have the general
structure shown in formula I:
##STR00002##
wherein X is, independently, O, N, or S;
Y is O, N, or S;
[0021] R.sup.1 is, independently, --H, --OH, --NH.sub.2,
--NO.sub.2, halogen, alkyl optionally substituted with --OH, or
alkoxy optionally substituted with --OH; and, R.sup.2 is --H, --OH,
--NH.sub.2, --C(O)NH.sub.2, alkyl, or alkoxy, any of which can be
optionally substituted with --OH, halogen, alkyl, or alkoxy;
R.sup.3 is --H, --OH, --NH.sub.2, --C(O)NH.sub.2, alkyl, or alkoxy,
any of which can be optionally substituted with --OH, halogen,
alkyl, or alkoxy; or an analog or a pharmaceutically acceptable
salt thereof.
[0022] In one embodiment, each X is N. In an exemplified
embodiment, Y is O. In another exemplified embodiment, each R.sup.1
is independently --OH or --CH.sub.2OH. In still another embodiment,
R.sup.2 is --NH.sub.2 optionally substituted with --OH. In a
further embodiment, R.sup.2 is --C(O)NH.sub.2.
[0023] In a specific embodiment, a compound of the invention has
the structure (formula II):
##STR00003##
[0024] Compounds of the invention inhibit not only highly activated
wild-type Akt resulting from alterations of upstream regulators
such as PTEN mutation but also constitutively active Akt mutants
including myr-AKT1, Myr-AKT2 and E40K-AKT1. A recent study
identified a recurring somatic mutation in PH domain of AKT1 in
human breast, colorectal and ovarian cancers that results in a
glutamic acid to lysine substitution at amino acid 17 (E17K) in the
lipid-binding pocket (Carpten et al. 2007). Lys 17 alters the
electrostatic interactions of the pocket and forms new hydrogen
bonds with a phosphoinositide ligand. This mutation activates AKT1
through pathological localization to the plasma membrane,
transforms cells and induces leukaemia in mice. Further, the E17K
substitution reduces the sensitivity to an allosteric kinase
inhibitor (Carpten. et al. 2007). As E40K-AKT1 mutant is similar to
E17K which can localize in the plasma membrane (Bellacosa et al.
1998), API-1 may also inhibit E17K-AKT1.
[0025] While compounds of the invention can be administered as
isolated compounds, these compounds can also be administered as
part of a pharmaceutical composition. The subject invention thus
further provides compositions in association with at least one
pharmaceutically acceptable carrier. The pharmaceutical composition
can be adapted for various routes of administration, such as
enteral, parenteral, intravenous, intramuscular, topical,
subcutaneous, and so forth. Administration can be continuous or at
distinct intervals, as can be determined by a person of ordinary
skill in the art.
[0026] The compounds of the invention can be formulated according
to known methods for preparing pharmaceutically useful
compositions. Formulations are described in a number of sources
which are well known and readily available to those skilled in the
art. For example, Remington's Pharmaceutical. Science (Martin 1995)
describes formulations which can be used in connection with the
subject invention. Formulations suitable for administration
include, for example, aqueous sterile injection solutions, which
may contain antioxidants, buffers, bacteriostats, and solutes that
render the formulation isotonic with the blood of the intended
recipient; and aqueous and nonaqueous sterile suspensions which may
include suspending agents and thickening agents. The formulations
may be presented in unit-dose or multi-dose containers, for example
sealed ampoules and vials, and may be stored in a freeze dried
(lyophilized) condition requiring only the condition of the sterile
liquid carrier, for example, water for injections, prior to use.
Extemporaneous injection solutions and suspensions may be prepared
from sterile powder, granules, tablets, etc. It should be
understood that in addition to the ingredients particularly
mentioned above, the compositions of the subject invention can
include other agents conventional in the art having regard to the
type of formulation in question.
[0027] As used herein, alkyl means straight or branched chain,
saturated or mono- or polyunsaturated hydrocarbon groups having
from 1 to 20 carbon atoms and C.sub.1-x alkyl means straight or
branched chain alkyl groups containing from one up to "X" number of
carbon atoms. For example, C.sub.1-6 alkyl means straight or
branched chain alkyl groups containing from one up to 6 carbon
atoms. Alkoxy means an alkyl-O-- group in which the alkyl group is
as previously described herein.
[0028] The term "halogen" means a halogen of the periodic table,
such as fluorine, chlorine, bromine, or iodine.
[0029] The term "optionally substituted" means optionally
substituted with one or more of the organic or inorganic groups
(e.g., alkyl, aryl, heteroaryl, acyl, alkenyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, or halogen), at
any available position or positions.
[0030] Examples of saturated alkyl groups include, but are not
limited to, methyl, ethyl, N-propyl, isopropyl, N-butyl,
tert-butyl, isobutyl, sec-butyl, N-pentyk N-hexyl, N-heptyl, and
N-octyl. An unsaturated alkyl group is one having one or more
double or triple bonds. Unsaturated alkyl groups include, for
example, ethenyl, propenyl, butenyl, hexenyl, vinyl, 2-propynyl,
2-isopentenyl, 2-butadienyl, ethynyl, 1-propynyl, 3-propynyl, and
3-butynyl. Specifically, alkyl can include, for example, methyl,
ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl,
3-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl,
tridecyl, tetradecyl, pentadecyl, vinyl, 1-propenyl, 2-propenyl,
1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl,
3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl,
5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl,
5-heptenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl,
6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl,
4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl;
1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl,
6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl,
1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl,
6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl,
11-dodecenyl, 1-tridecenyl, 2-tridecenyl, 3-tridecenyl,
4-tridecenyl, 5-tridecenyl, 6-tridecenyl, 7-tridecenyl,
8-trideeenyl, 9-triclecenyl, 10-tridecenyl, 11-tridecenyl,
12-tridecenyl, 1-tetradecenyl, 2-tetradeeenyl, 3-tetradecenyl,
4-tetradecenyl, 5-tetradecenyl, 6-tetradecenyl, 7-tetrad cc enyl,
8-tetradecenyl, 9-tetradecenyl, 10-tetradecenyl, 11-tetradecenyl,
12-tetradecenyl, 13-tetradeceny, 1-pentadecenyl, 2-pentadecenyl,
3-pentadecenyl, 4-pcntadecenyl, 5-pentadecenyl, 6-pentadecenyl,
7-pentadecenyl, 8-pentadecenyl, 9-pentadecenyl, 10-pentadecenyl,
11-pentadecenyl, 12-pentadecenyl, 13-pentadecenyl, or
14-pentadecenyl; "alkoxy" can include methoxy, ethoxy, propoxy,
isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy,
hexoxy, heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy,
dodecyloxy, tridecyloxy, tetradecyloxy, or pentadecyloxy.
[0031] The compounds of the present invention include all hydrates
and salts that can be prepared by those of skill in the art. Under
conditions where the compounds of the present invention are
sufficiently basic or acidic to form stable nontoxic acid or base
salts, administration of the compounds as salts may be appropriate.
Examples of pharmaceutically acceptable salts are organic acid
addition salts formed with acids that form a physiological
acceptable anion, for example, tosylate, methanesulfonate, acetate,
citrate, malonate, tartarate, succinate, benzoate, ascorbate,
alpha-ketoglutarate, and alpha-glycerophosphate. Suitable inorganic
salts may also be formed, including hydrochloride, sulfate,
nitrate, bicarbonate, and carbonate salts.
[0032] Pharmaceutically acceptable salts of a compound may be
obtained using standard procedures well known in the art, for
example, by reacting a sufficiently basic compound such as an amine
with a suitable acid affording a physiologically acceptable anion.
Alkali metal (for example, sodium, potassium or lithium) or
alkaline earth metal (for example calcium) salts of carboxylic
acids can also be made.
[0033] As used herein, the term "analogs" refers to compounds which
are substantially the same as another compound but which may have
been modified by, for example, adding side groups, oxidation or
reduction of the parent structure. Analogs of compounds of the
invention can be readily prepared using commonly known standard
reactions. These standard reactions include, but are not limited
to, hydrogenation, alkylation, acetylation, and acidification
reactions. Chemical modifications can be accomplished by those
skilled in the art by protecting all functional groups present in
the molecule and deprotecting them after carrying out the desired
reactions using standard procedures known in the scientific
literature (Greene et al. 1999; Honda et al. 1997; Honda et al.
1998; Konoike et al. 1997; Honda et al. 2000; each of which are
hereby incorporated herein by reference in their entirety). Analogs
exhibiting the desired biological activity (such as induction of
apoptosis, cytotoxicity, cytostaticity, induction of cell cycle
arrest, anti-angiogenic properties, etc.) can be identified or
confirmed using cellular assays or other in vitro or in vivo
assays.
[0034] It will be appreciated that compounds of the invention can
contain one or more asymmetrically substituted carbon atoms (i.e.,
carbon centers). The presence of one or more of the asymmetric
centers in a compound of the invention, can give rise to
stereoisomers, and in each case, the invention is to be understood
to extend to all such stereoisomers, including enantiomers and
diastereomers, and mixtures including racemic mixtures thereof.
[0035] Compounds and compositions of the invention are useful for
various non-therapeutic and therapeutic purposes. The compounds and
compositions may be used for reducing aberrant cell growth in
animals and humans. Because of such anti-proliferative properties
of the compounds, they are uSeful in reducing unwanted cell growth
in a wide variety of settings including in vitro and in vivo.
[0036] Therapeutic application of compounds and compositions
containing them can be accomplished by any suitable therapeutic
method and technique presently or prospectively known to those
skilled in the art. Further, compounds of the invention have use as
starting materials or intermediates for the preparation of other
useful compounds and compositions.
[0037] Compounds of the invention, and compositions thereof, may be
locally administered at one or more anatomical sites, such as sites
of unwanted cell growth (such as a tumor site or benign skin
growth, e.g., injected or topically applied to the tumor or skin
growth) or sites of fungal infection, optionally in combination
with a pharmaceutically acceptable carrier such as an inert
diluent. Compounds of the invention, and compositions thereof, may
be systemically administered, such as intravenously or orally,
optionally in combination with a pharmaceutically acceptable
carrier such as an inert diluent, or an assimilable edible carrier
for oral delivery. They may be enclosed in hard or soft shell
gelatin capsules, may be compressed into tablets, or may be
incorporated directly with the food of the patient's diet. For oral
therapeutic administration, the active compound may be combined
with one or more excipients and used in the form of ingestible
tablets, buccal tablets, troches, capsules, elixirs, suspensions,
syrups, wafers, aerosol sprays, and the like.
[0038] The tablets, troches, pills, capsules, and the like may also
contain the following: binders such as gum tragacanth, acacia, corn
starch or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent such as corn starch, potato starch, alginic
acid and the like; a lubricant such as magnesium stearate; and a
sweetening agent such as sucrose, fructose, lactose or aspartame or
a flavoring agent such as peppermint, oil of wintergreen, or cherry
flavoring may be added. When the unit dosage form is a capsule, it
may contain, in addition to materials of the above type, a liquid
carrier, such as a vegetable oil or a polyethylene glycol. Various
other materials may be present as coatings or to otherwise modify
the physical form of the solid unit dosage form. For instance,
tablets, pills, or capsules may be coated with gelatin, wax,
shellac, or sugar and the like. A syrup or elixir may contain the
active compound, sucrose or fructose as a sweetening agent, methyl
and propylparabens as preservatives, a dye and flavoring such as
cherry or orange flavor. Of course, any material used in preparing
any unit dosage form should be pharmaceutically acceptable and
substantially non-toxic in the amounts employed. In addition, the
active compound may be incorporated into sustained-release
preparations and devices.
[0039] Compounds and compositions of the invention, including
pharmaceutically acceptable salts or analogs thereof, can be
administered intravenously, intramuscularly, or intraperitoneally
by infusion or injection. Solutions of the active agent or its
salts can be prepared in water, optionally mixed with a nontoxic
surfactant. Dispersions can also be prepared in glycerol, liquid
polyethylene glycols, triacetin, and mixtures thereof and in oils.
Under ordinary conditions of storage and use, these preparations
can contain a preservative to prevent the growth of
microorganisms.
[0040] The pharmaceutical dosage forms suitable for injection or
infusion can include sterile aqueous solutions or dispersions or
sterile powders comprising the active ingredient which are adapted
for the extemporaneous preparation of sterile injectable or
infusible solutions or dispersions, optionally encapsulated in
liposomes. The ultimate dosage form should be sterile, fluid and
stable under the conditions of manufacture and storage. The liquid
carrier or vehicle can be a solvent or liquid dispersion medium
comprising, for example, water, ethanol, a polyol (for example,
glycerol, propylene glycol, liquid polyethylene glycols, and the
like), vegetable oils, nontoxic glyceryl esters, and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the formation of liposomes, by the maintenance of the
required particle size in the case of dispersions or by the use of
surfactants. Optionally, the prevention of the action of
microorganisms can be brought about by various other antibacterial
and antifungal agents, for example, parabens, chlorobutanol,
phenol, sorbic acid, thimerosal, and the like. In many cases, it
will be preferable to include isotonic agents, for example, sugars,
buffers or sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the inclusion of agents that
delay absorption, for example, aluminum monostearate and
gelatin.
[0041] Sterile injectable solutions are prepared by incorporating a
compound of the invention in the required amount in the appropriate
solvent with various other ingredients enumerated above, as
required, followed by filter sterilization. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and the freeze
drying techniques, which yield a powder of the active ingredient
plus any additional desired ingredient present in the previously
sterile-filtered solutions.
[0042] For topical administration, compounds of the invention may
be applied in as a liquid or solid. However, it will generally be
desirable to administer them topically to the skin as compositions,
in combination with a dermatologically acceptable carrier, which
may be a solid or a liquid. Compounds and compositions of the
subject invention can be applied topically to a subject's skin to
reduce the size (and may include complete removal) of malignant or
benign growths, or to treat an infection site. Compounds of the
invention can be applied directly to the growth or infection site.
Preferably, the compounds are applied to the growth or infection
site in a formulation such as an ointment, cream, lotion, solution,
tincture, or the like. Drug delivery systems for delivery of
pharmacological substances to dermal lesions can also be used, such
as that described in U.S. Pat. No. 5,167,649.
[0043] Useful solid carriers include finely divided solids such as
talc, clay, microcrystalline cellulose, silica, alumina and the
like. Useful liquid carriers include water, alcohols or glycols or
water-alcohol/glycol blends, in which the compounds can be
dissolved or dispersed at effective levels, optionally with the aid
of non-toxic surfactants. Adjuvants such as fragrances and
additional antimicrobial agents can be added to optimize the
properties for a given use. The resultant liquid compositions can
be applied from absorbent pads, used to impregnate bandages and
other dressings, or sprayed onto the affected area using pump-type
or aerosol sprayers, for example.
[0044] Thickeners such as synthetic polymers, fatty acids, fatty
acid salts and esters, fatty alcohols, modified celiuloses or
modified mineral materials can also be employed with liquid
carriers to form spreadable pastes, gels, ointments, soaps, and the
like, for application directly to the skin of the user. Examples of
useful dermatological compositions which can be used to deliver a
compound to the skin are disclosed in U.S. Pat. No. 4,608,392; U.S.
Pat. No. 4,992,478; U.S. Pat. No. 4,559,157; and U.S. Pat. No.
4,820,508.
[0045] Useful dosages of the compounds and pharmaceutical
compositions of the present invention can be determined by
comparing their in vitro activity, and in vivo activity in animal
models. Methods for the extrapolation of effective dosages in mice,
and other animals, to humans are known to the art; for example, see
U.S. Pat. No. 4,938,949.
[0046] The present invention also concerns pharmaceutical
compositions comprising a compound of the invention in combination
with a pharmaceutically acceptable carrier. Pharmaceutical
compositions adapted for oral, topical or parenteral
administration, comprising an amount of a compound constitute a
preferred embodiment of the invention. The dose administered to a
patient, particularly a human, in the context of the present
invention should be sufficient to achieve a therapeutic response in
the patient over a reasonable time frame, without lethal toxicity,
and preferably causing no more than an acceptable level of side
effects or morbidity. One skilled in the art will recognize that
dosage will depend upon a variety of factors including the
condition (health) of the subject, the body weight of the subject,
kind of concurrent treatment, if any, frequency of treatment,
therapeutic ratio, as well as the severity and stage of the
pathological condition.
[0047] The subject invention also concerns methods for inhibiting
the survival or proliferation or killing a cancer or tumor cell or
other cell in which expression of an Akt protein is elevated or
constitutively active, comprising contacting the cell with an
effective amount of a compound of formula I, or a salt or analog
thereof. In a specific embodiment, the compound has the structure
shown in formula II, or a salt or analog thereof. In one
embodiment, the cell is a human cell or other mammalian cell.
Cancer cells that can be inhibited or killed using the subject
methods include those cells that are metastatic in nature. Thus,
inhibition of metastasis of a cancer or tumor cell is also
contemplated by the present invention. The methods can be practiced
in vitro or in vivo.
[0048] The subject invention also concerns methods for treating
oncological disorders, such as cancer or a tumor in a person or
animal comprising administering an effective amount of a compound
of formula I, or a salt or analog thereof, to the person or animal.
In a specific embodiment, a compound has the formula shown in
formula II, or a salt or analog thereof. In one embodiment, an
effective amount of a compound or composition of the present
invention is administered to a patient having an oncological
disorder and who is in need of treatment thereof. The inhibitor can
be administered prior to, subsequent to, or in conjunction with
chemotherapy, immunotherapy and/or radiotherapy. Methods of the
invention can optionally include identifying a patient who is or
may be in need of treatment of an ontological disorder. Patients in
need of treatment using the methods of the present invention can be
identified using standard techniques known to those in the medical
or veterinary professions, as appropriate. In one embodiment, the
patient can be a human or other mammal, such as a primate (monkey,
chimpanzee, ape, etc.), dog, cat, cow, pig, or horse, or other
animals having an ontological disorder. Means for administering and
formulating compounds of the invention for administration to a
patient are known in the art, examples of which are described
herein. Ontological disorders within the scope of the invention
include, but are not limited to, cancer and/or tumors of the anus,
bile duct, bladder, bone, bone marrow, bowel (including colon and
rectum), breast, eye, gall bladder, kidney, mouth, larynx,
esophagus, stomach, testis, cervix, head, neck, ovary, lung,
mesothelioma, neuroendocrine, penis, skin, spinal cord, thyroid,
vagina, vulva, uterus, liver, muscle, pancreas, prostate, blood
cells (including lymphocytes and other immune system cells), and
brain. Specific cancers contemplated for treatment with the present
invention include carcinomas, Karposi's sarcoma, melanoma,
mesothelioma soft tissue sarcoma, leukemia (acute lymphoblastic,
acute myeloid, chronic lymphocytic, chronic myeloid, and other),
and lymphoma (Hodgkin's and non-Hodgkin's), and multiple
myeloma.
[0049] For the treatment of ontological disorders, compounds and
compositions contemplated by the present invention can be
administered to a patient in need of treatment prior to, subsequent
to, or in combination with other antitumor or anticancer agents or
substances (e.g., chemotherapeutic agents, immunotherapeutic
agents, radiotherapeutic agents, etc.) and/or with radiation
therapy and/or with surgical treatment to remove a tumor. For
example, compounds and compositions of the present invention can be
used in methods of treating cancer wherein the patient is to be
treated or is or has been treated with mitotic inhibitors such as
taxol or vinblastine, alkylating agents such as cyclophosamide or
ifosfamide, antimetabolites such as 5-fluorouracil or hydroxyurea,
DNA intercalators such as adriamycin or bleomycin, topoisomerase
inhibitors such as etoposide or camptothecin, antiangiogenic agents
such as angiostatin, antiestrogens such as tamoxifen, and/or other
anti-cancer drugs or antibodies, such as, for example, GLEEVEC
(Novartis Phan iaceuticals Corporation) and HERCEPTIN (Genentech,
Inc.), respectively. These other substances or radiation treatments
may be given at the same as or at different times from the
compounds of this invention. Examples of other chemotherapeutic
agents contemplated within the scope of the invention include, but
are not limited to, altretamine, bleomycin, bortezomib (VELCADE),
busulphan, calcium folinate, capecitabine, carboplatin, carmustine,
chlorambucil, cisplatin, cladribine, crisantaspase,
cyclophosphamide, cytarabine, dacarbazine, dactinomycin,
daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide,
fludarabine, fluorouracil, gefitinib (IRESSA), gemeitabine,
hydroxyurea, idarubicin, ifosfamide, imatinib (GLEEVEC),
irinotecan, liposomal doxorubicin, lomustine, melphalan,
mercaptopurine, methotrexate, mitomycin, mitoxantrone, oxaliplatin,
paclitaxel, pentostatin, procarbazine, raltitrexed, streptozocin,
tegafur-uracil, temozolomide, thiotepa, tioguanine/thioguanine,
topotecan, treosulfan, vinblastine, vincristine, vindesine,
vinorelbine. In an exemplified embodiment, the chemotherapeutic
agent is melphalan. Examples of immunotherapeutic agents
contemplated within the scope of the invention include, but are not
limited to, alemtuzumab, cetuximab (ERBITUX), gemtuzumab, iodine
131 tositumomab, rituximab, trastuzamab (HERCEPTIN). The subject
invention also concerns methods for treating an oncological
disorder comprising administering an effective amount of a compound
of the invention prior to, subsequent to, and/or in combination
with administration of a chemotherapeutic agent, an
immunotherapeutic agent, a radiotherapeutic agent, or
radiotherapy.
[0050] The subject invention also concerns methods for inhibiting
the Akt/PKB pathway in a cell by contacting the cell with an
effective amount of a compound or composition of the invention. In
one embodiment, the compound binds to and inhibits the activity of
an Akt1, AKT2, and/or AKT3 protein. In a specific embodiment, the
compound has the structure shown in formula II, or a salt or analog
thereof. In one embodiment, the cell is a human or mammalian cell,
and can be a cancer or tumor cell or other cell that exhibits
abnormal proliferation, survival, migration or differentiation. In
one embodiment, the cell constitutively expresses or expresses
elevated or abnormal levels of an Akt protein, such as Akt1, AKT2,
and/or AKT3.
[0051] The subject invention also concerns methods for treating a
person or animal having a disorder associated with constitutive,
abnormal, or elevated expression of an Aid protein in a cell,
wherein a therapeutically effective amount of a compound or
composition of the invention is administered to the person or
animal. The disorder can be one characterized, for example, by
abnormal cell proliferation, cell survival, cell migration, and/or
cell differentiation. In one embodiment, the compound binds to and
inhibits activity of an Akt1, AKT2, and/or AKT3 protein. In a
specific embodiment, the compound has the structure shown in
formula II, or a salt or analog thereof.
[0052] Depending upon the disorder or disease condition to be
treated, a suitable dose(s) may be that amount that will reduce
proliferation or growth of the target cell(s). In the context of
cancer, a suitable dose(s) is that which will result in a
concentration of the active agent in cancer tissue, such as a
malignant tumor, which is known to achieve the desired response.
The preferred dosage is the amount which results in maximum
inhibition of cancer cell growth, without unmanageable side
effects. Administration of a compound can be continuous or at
distinct intervals, as can be determined by a person of ordinary
skill in the art.
[0053] To provide for the administration of such dosages for the
desired therapeutic treatment, in some embodiments, pharmaceutical
compositions of the invention can comprise between about 0.1% and
45%, and especially, 1 and 15%, by weight of the total of one or
more of the compounds based on the weight of the total composition
including carrier or diluents. Illustratively, dosage levels of the
administered active ingredients can be: intravenous, 0.01 to about
20 mg/kg; intraperitoneal, 0.01 to about 100 mg/kg; subcutaneous,
0.01 to about 100 mg/kg; intramuscular, 0.01 to about 100 mg/kg;
orally 0.01 to about 200 mg/kg, and preferably about 1 to 100
mg/kg; intranasal instillation, 0.01 to about 20 mg/kg; and
aerosol, 0.01 to about 20 mg/kg of animal (body) weight.
[0054] The subject invention also concerns kits comprising one or
more compounds of the invention, or a composition comprising a
compound of the invention, or an analog or salt of the foregoing,
in one or more containers. In one embodiment, the kit comprises a
compound of formula II, or a pharmaceutically acceptable salt or
analog thereof. Kits of the invention can optionally include
pharmaceutically acceptable carriers and/or diluents. In one
embodiment, a kit of the invention includes one or more other
components, adjuncts, or adjuvants as described herein. In another
embodiment, a kit includes one or more anti-cancer agents, such as
those agents described herein. In one embodiment, a kit of the
invention includes instructions or packaging materials that
describe how to administer a compound or composition of the kit.
Containers of the kit can be of any suitable material, e.g., glass,
plastic, metal, etc., and of any suitable size, shape, or
configuration. In one embodiment, a compound of the invention is
provided in the kit as a solid, such as a tablet, pill, or powder
form. In another embodiment, a compound of the invention is
provided in the kit as a liquid or solution. In one embodiment, the
kit comprises an ampoule or syringe containing a compound of the
invention in liquid or solution form.
[0055] Mammalian species which benefit from the disclosed methods
include, but are not limited to, primates, such as apes,
chimpanzees, orangutans, humans, monkeys; domesticated animals
(e.g., pets) such as dogs, cats, guinea pigs, hamsters, Vietnamese
pot-bellied pigs, rabbits, and ferrets; domesticated farm animals
such as cows, buffalo, bison, horses, donkey, swine, sheep, and
goats; exotic animals typically found in zoos, such as bear, lions,
tigers, panthers, elephants, hippopotamus, rhinoceros, giraffes,
antelopes, sloth, gazelles, zebras, wildebeests, prairie dogs,
koala bears, kangaroo, opossums, raccoons, pandas, hyena, seals,
sea lions, elephant seals, otters, porpoises, dolphins and whales.
Other species that may benefit from the disclosed methods include
fish, amphibians, avians, and reptiles. As used herein, the terms
"patient" and "subject" are used interchangeably and are intended
to include such human and non-human species. Likewise, in vitro
methods of the present invention can be carried out on cells of
such human and non-human species.
Materials and Methods
[0056] Cell Lines and NCI Diversity Set.
[0057] All cell lines used in this study were either purchased from
ATCC or described previously (Cheng et al. 1997; Jiang et al. 2000;
Yang et al. 2004; Yang et al. 2005). The NCI Structural Diversity
Set is a library of 1,992 compounds selected front the
approximately 140,000-compound NCl drug depository. In-depth data
on the selection, structures, and activities of these diversity set
compounds can be found on the NCl Developmental Therapeutics
Program web site (Jones et al. 1991).
[0058] Screening for Inhibition of Akt-transformed Cell Growth.
[0059] AKT2 transformed NIH3T3 cells or LXSN vector-transfected
NIH3T3 control cells (Cheng et al. 1997) were plated into 96-well
tissue culture plate. Following treatment with 5 .mu.M of NCI
Diversity Set compound, cell growth was detected with CellTier 96
One Solution Cell Proliferation kit (Promega). Compounds that
inhibit growth in AKT2-transformed but not LXSN-transfected NIH3T3
cells were considered as candidates of Akt inhibitor and subjected
to further analysis.
[0060] In vitro Protein Kinase Cell Survival and Apotosis
Assays.
[0061] In vitro kinase was performed as previously described (Jiang
et al. 2000). Cell survival was assayed with MTT (Sigma). Apoptosis
was detected with annexin V (BD Biosciences), which was performed
according to manufacture's instruction. Recombinant Aid and PDK1
were purchased from Upstate Biotechnology Inc.
[0062] Antitumor Activity in the Nude Mouse Tumor Xenograft
Model.
[0063] Tumor cells were harvested, resuspended in PBS, and injected
subcutaneous (s.c.) into the right and left flanks
(2.times.10.sup.6 cells/flank) of 8-week-old female nude mice as
reported previously (Sun et al. 1999). When tumors reached about
100-150 mm.sup.3, animals were randomized and dosed intraperitoneal
(i.p.) with vehicle or drug daily. Control animals received
dimethylsulfoxide (DMSO) (20%) vehicle and treated animals were
injected with API-1 (10 mg/kg/day) in 20% DMSO.
[0064] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety, including all figures and tables, to
the extent they are not inconsistent with the explicit teachings of
this specification.
[0065] Following are examples that illustrate procedures for
practicing the invention. These examples should not be construed as
limiting. All percentages are by weight and all solvent mixture
proportions are by volume unless otherwise noted.
Example 1
Identification of Small Molecule Akt/PKB Pathway Inhibitor-1,
API-1, by Screening NCI Diversity Set
[0066] Due to the fact that aberrant activation of Akt pathway
occurs in almost 50% all the human malignancy and inhibition of Akt
induces cell growth arrest and apoptosis, it has drawn interest
from industry and academia to develop small molecule Akt inhibitor
for anti-cancer drug discovery (Cheng et al. 2005; Granville et al.
2006). While a dozen Akt inhibitors have been reported, many of
them lack anti-tumor activity in vivo. A lipid-based Akt inhibitor,
perifosinc, has been reported in phase I and II studies (Van
Ummersen et al. 2004; Bailey et al. 2006). However, in neither
study was modulation of Akt assessed. A recent phase II study of
perifosine in pancreatic cancer was terminated as a result of
unacceptable adverse events during the first stage (Marsh et al.
2007). Nevertheless, there is a need to develop potent selective
Akt inhibitors that are void of inhibiting other kinase activities
with minimal adverse effect. To identify small molecule
inhibitor(s) of Akt, we have evaluated a chemical library of
1,992-compounds from the NCI (the NCI Diversity Set) for agents
capable of inhibition of growth in AKT2-transformed but not empty
vector LXSN-transfected NIH3T3 cells as described in "Materials and
Methods". Triple experiments showed that 32 compounds inhibited
growth only in AKT2-transformed cells. We previously characterized
one of them, named API-2/triciribine that is a pan-Akt inhibitor
with anti-tumor activity in vitro and in vivo and currently in
phase I clinic trail (Yang et al. 2004).
[0067] In the present study, we showed that Akt/PKB inhibitor-1
(API-1) specifically inhibits kinase activity and phosphorylation
levels of Akt in living cells. FIG. 1A shows the chemical structure
of API-1 (NSC 177223), which has no chemical name and has not been
tested in NCI 60 cell lines (http://dtp.nci.nih.gov/). Since API-1
inhibited selectively AKT2 transformed cells over untransformed
parental cells, we first examined whether API-1 is an inhibitor of
AKT2 kinase and whether it also inhibits other two members of Akt.
HEK293 cells were transfected with HA-tagged wild type Akt1, AKT2
and AKT3. Following serum starvation for overnight, cells were
treated with API-1 for 60 min prior to EGF stimulation and
immunoprecipitated with anti-HA antibody. The immunoprecipitates
were subjected to in vitro kinase assay. FIG. 1B shows that API-1
suppressed insulin-induced kinase activity of Akt1, AKT2 and AKT3.
We next examine if API-1 inhibits Akt in living calls. OVCAR3
cells, which express elevated levels of phosphor-Akt, were treated
with different doses of API-1 for 3 hours. Immunoblotting analysis
with anti-phospho-Akt-S473 antibody showed that API-1 efficiently
reduced phosphorylation level of Akt and IC.sub.50 is approximately
at 0.8 .mu.M. However, total Akt levels were no change (FIG. 1C).
Further, we examined if API-1 directly inhibits Akt kinase activity
in vitro. Recombinant constitutively active Akt protein was
incubated with Akt/SGK substrate peptide (Upstate) in a kinase
buffer containing different amounts of API-1 and compound E, a
pan-kinase ATP-competitor inhibitor including Akt, as positive
control. Triple experiments showed no effect of APT-1 on Akt kinase
activity (FIG. 1D), suggesting that API-1 does not directly inhibit
Akt in vitro and that API-1 functions neither as ATP nor substrate
competitor.
Example 2
API-1 Does Not Inhibit Upstream Activators of Akt
[0068] Akt is activated by extracellular stimuli and intracellular
signal molecules through a PI3K-dependent manner, which is
negatively regulated by PTEN. Activation of PI3K or mutation of
PTEN will activate PDK1 leading to induction of Akt kinase
activity. Therefore, API-1 inhibition of Akt could result from
targeting upstream molecule(s) of Akt, such as PI3K and PDK1. To
this end, we next examined if API-1 inhibits PI3K and/or PDK1.
HEK293 cells were serum-starved and then treated with API-1 or PI3K
inhibitor, wortmannin, for 1 hour prior to EGF stimulation. PI3K
was immunoprecipitated with anti-p110a antibody. The
immunoprecipitates were subjected to in vitro PI3K kinase assay
using PI-4-P as a substrate. As shown in FIG. 2A, the EGF-induced.
PI3K activity was inhibited by wortmannin but not by API-1. To
evaluate the effect of API-1 on PDK1, we performed in vitro PDK1
kinase assay using SGK kinase as readout (Upstate Biotechnology
Inc.). Unlike PDK1 inhibitor UCN-01 (Sato et al. 2002), API-1 had
no effect on in vitro PDK1 kinase activity (FIG. 2B). To further
evaluate the effect of API-1 on PDK1 activation in living cells, we
examined phosphorylation level of PDK1-Ser241, a residue that is
autophosphorylated and is critical for its activity (Casamayor et
al. 1999), following API-1 treatment of OVCAR3 cells. FIG. 2D shows
that phosphorylation levels or PDK1 were not inhibited by
API-1.
Example 3
API-1 Is Highly Selective for the Akt Over AGC kinase Members PKA,
PKC and SGK, and Other Signaling Molecules ERK, JNK, p38, and
STAT3
[0069] Akt belongs to AGC(PKA/PKG/PKC) kinase family, which also
include PKA, PKC, serum- and glucocorticoid-inducible kinase (SGK),
p90 ribosomal S6 kinase, p70.sup.S6K, mitogen- and stress-activated
protein kinase and PKC-related kinase. Among AGC kinase family,
protein structures of PKA, PKC and SGK are much closer to Akt
kinase than other members. Therefore, we next examined the effects
of API-1 on the enzymatic activities of these 3 kinases. In vitro
PKA kinase assay and SGK kinase assay was performed by
pre-incubated indicated dose of API-1 or specific inhibitor with
recombinant PKA or SGK protein for 30 minutes before starting
kinase assay by adding kemptide or Akt/SGK substrate peptide and
[.gamma.-.sup.32P]ATP in kinase buffer. In vitro kinase assay
showed that the kinase activities of PKA and SGK were inhibited by
PKAT and compound E respectively, whereas API-1 exhibited no effect
on their, activities (FIGS. 2C and 2E). To further evaluate the
effect of API-1 on the PKA and PKCu activation in living cells,
OVCAR3 cells were treated with indicated doses of API-1 or specific
inhibitor of PKA and PKC, immunoblotting analysis showed that
phosphorylation levels of PKA and PKC were not inhibited by API-1
(FIG. 2D). In addition, HEK293 cells were transfected with
HA-tagged SGK. In vitro kinase assay showed that EGF-induced SGK
kinase activity was attenuated by wortmannin but not API-1 (FIG.
2F).
[0070] To determine whether API-1 has effect on other oncogenic
survival pathways, OVCAR3 cells were treated with API-1 (10
.alpha.M) for different times and immanoblotted with commercially
available anti-phospho-antibodies. We did not observe the
detectable changes of phosphorylation levels of Stat3, JNK, p38 and
Erk1/2 after API-1 treatment (FIG. 2G). These data indicate that
API-1 could specifically inhibit the Akt pathway.
Example 4
API-1 inhibits Constitutively Active Akt and its Downstream
Targets
[0071] Since API-1 could not directly inhibit Akt in vitro but
abrogated kinase activity and phosphorylation of Akt in the living
cells without effect on PI3K and PDK1, this led us to assume that
this compound may interact with Akt protein, but not ATP-binding
site, to prevent it from phosphorylation of Thr308 and Ser473 by
PDK1 and PDK2. If this is authentic, API-1 should also inhibit
activation of constitutively active Akt since its activation still
requires phosphorylation of Thr308 and Ser473 (Sun et al. 2001). To
test this hypothesis, HEK293 cells were transfected with HA-tagged
Myr-Akt1-Akt2 and -Akt1E40K. Following serum starvation overnight,
cells were treated with or without API-1. Myr-Akt1, Myr-Akt2 and
Akt1-E40K were immunoprecipitated with anti-HA antibody. The
immunoprecipitates were subjected to in vitro kinase assay and
immunoblotting analysis with anti-pliospho-Akt-T308 antibody. FIG.
3A shows that in vitro Akt kinase activity as well as
phosphorylation levels of Myr-Aka, Myr-Akt2 and Akt1-E40K were
inhibited by API-1, supporting the notion that API-1 might bind to
Akt molecule to prevent it from activation within the cells.
[0072] Akt exerts its cellular function through phosphorylation of
a number of proteins (Datta et al. 1999). We next examined whether
API-1 inhibits downstream targets of Akt. Since GSK3f3 and mTOR are
two of the major Akt targets, we evaluated the effects of API-1 on
phosphorylation levels of GSK.beta. and S6, a substrate of
p70.sup.S6K. Following treatment of OVCAR3 with AI-1,
immunoblotting analysis revealed that API-1 largely inhibited their
phosphorylation (FIG. 3B).
Example 5
API-1 Suppresses Cell Growth and Induces Apoptosis in
Akt-Overexpressing Cancer Cell Lines
[0073] Akt is a major pro-growth and pro-survival pathway. Cancer
cells with elevated Akt kinase exhibit more resistant to
chemotherapeutic drug-induced cell growth arrest and cell death
whereas knockdown Akt sensitizes to apoptosis induced by different
proapoptotic stimuli (Solit et al. 2003; Xu et al. 2003; Jetzt et
al. 2003). The ability of API-1 to selectively inhibit the Akt
pathway suggests that it should inhibit proliferation and/or
induces apoptosis preferentially in those tumor cells with aberrant
expression/activation of Akt. To test this, API-1 was used to treat
the cells that express constitutively active Akt, caused by
overexpression of Akt (OVCAR3, OVCAR8, MCF7 and PANC1) or mutations
of the PTEN gene (MDA-MB-468PC-3 and LNCaP), and cells that do not
(OVCAR5, MDA-MB-435s, DU-145 and COL0357). Immunoblotting analysis
showed that phosphorylation levels of Akt were significantly
inhibited by API-1 in the cells expressing elevated Akt while
phospho-Akt was also reduced by API-1 in the cell lines exhibiting
low levels of Akt (FIG. 4A and data not shown). However, API-1
induces PARP cleavage and inhibits cell growth in a much higher
degree in Akt-overexpressing/activating cells as compared to those
with low levels of Akt (FIGS. 4A, 4B, and 4C). API-1 treatment
inhibited cell proliferation by approximate 50-70% in
Akt-overexpressing/activating cell lines, OVCAR3, OVCA8, MDA-MB-468
and MCF7, whereas only by about 10-30% in OVCAR5, and MDA-MB-435s
cells (FIGS. 4B and 4C). Moreover, API-1 induces apoptosis by
9-fold and 4.6-fold in OVACAR3 and MDA-MB-468, respectively,
whereas much less apoptosis was observed in API-1-treated OVCAR5
and MDA-MB-435s cells (FIGS. 4D-4G). Thus, API-1 inhibits cell
growth and induces apoptosis preferentially in the cells that
express aberrant Aid.
Example 6
API-1 Inhibits the Growth of Tumors in Nude Mice that Overexpress
Akt
[0074] It has previously been shown that aberrant activation and
overexpression of Akt are frequently detected in human ovarian and
pancreatic cancer (Cheng et al. 1992) and that antisense of Akt
significantly inhibits tumor cell growth and invasion (Cheng at al.
1996). Further, inhibition of Akt pathway by inhibitors of PI3K,
HSP70, Src and farnesyltransferase resulted in cell growth arrest
and induction of apoptosis (Solit et al. 2003; Xu at al. 2003).
Because API-1 inhibits Akt signaling and induces apoptosis and cell
growth arrest in cancer cells with elevated levels of Akt (FIGS.
4A-4G), it was reasoned that the growth oftumors with elevated
levels of Akt should be more sensitive to API-1 than that of tumors
with low levels of Akt in nude mice. To this end,
Akt-overexpressing cells (OVCAR3 and PANC-1) were s.c. implanted
into the left flank, and those cell lines that express low levels
of Akt (OVCAR5 and COLO357) were s.c. implanted into the right
flank of mice. When the tumors reached an average size of about
100-150 mm.sup.3, the animals were randomized and treated i.p. with
either vehicle or API-1 (10 mg/kg/day). As illustrated in FIGS.
5A-5J, OVCAR3 and PANC1 tumors treated with vehicle control
continued to grow. API-1 inhibited OVCAR3 and PANC1 tumor growth by
70% and 50%, respectively (FIGS. 5C-5F and 5G-5J). In contrast,
APT-1 had little effect on the growth of OVCAR5 and COL0357 cells
in nude mice (FIGS. 5A-5J). At dose 10 mg/kg/day, API-1 had no
effects on blood glucose level, body weight, activity and food
intake of mice (data not shown). In treated tumor samples,
phosphorylation levels of Akt were reduced by API-1 about 70%
without change of total Akt content (FIGS. 5K-5L). Taken together,
these results indicate that API-1 selectively inhibits the growth
of tumors with elevated levels of Akt.
[0075] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application and the scope of the
appended claims. In addition, any elements or limitations of any
invention or embodiment thereof disclosed herein can be combined
with any and/or all other elements or limitations (individually or
in any combination) or any other invention or embodiment thereof
disclosed herein, and all such combinations are contemplated with
the scope of the invention without limitation thereto.
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